1. Field of the Invention
This invention relates to novel hollow porous microspheres having the pores thereof closed, e.g. coated, impregnated, sealed, filled or covered with a fluid permeable substance, and to the use of these microspheres for providing a non-rupturable support and protective environment for a biocatalytic substance, e.g. microorganisms, cells, cellular products, enzymes, antibodies, and other biochemically active materials, and to the use of the encapsulated biocatalysts, as bioreactors in biochemical processes.
More particularly, the invention relates to bioreactors, i.e. encapsulated biocatalysts, such as, for example, enzymes, antibodies, hormones, interferons, lymphokines, and other non-living biologically active substances, as well as various living substances, including viruses, bacteria, yeasts, single cells, organelles, cellular masses, tissues, and the like, wherein the encapsulating containers are in the form of non-rupturable hollow porous microspheres wherein the pores are coated, impregnated, sealed, filled, covered, or otherwise closed-off with a fluid permeable gel and/or permselective membrane material; and to the biochemical processes using the novel bioreactors.
2. Discussion of the Prior Art
A bioreactor may be broadly defined as a biocatalyst in a container. The biocatalyst may be any one of a wide variety of chemically active biological substances, such as, for example, enzymes, hormones, antibodies, interferons, lymphokines, and other non-living substances, and living microorganisms, such as bacteria, viruses, yeast, single cells, organelles, cellular masses, tissues, etc.
Traditionally, bioreactors have taken the form of fermentation vats, as in the production of alcohols, sugars and other fermentation products. Recently, as the containers or supports for these biocatalysts porous solid and porous hollow organic and inorganic fibers, and microcapsules have been proposed and have been commercially available. The porous fibers and microcapsules provide the advantage of large surface areas for a small volume and when the biocatalyst is encapsulated in the fiber or microcapsule, a sterile environment. A general discussion relating to the use of bioreactors in biotechnological processes can be found in Science, Vol. 219, Feb. 11, 1983, pp. 728-733, "Bioreactors: Design and Operation," Charles L. Cooney.
As representative of the U.S. Patent art relating to hollow fiber membrane cell culture devices mention can be made of the following: U.S. Pat. Nos. 3,821,087, 3,883,393, 3,997,396, 4,087,327, 4,184,922, 4,189,534, 4,200,689, 4,201,845, 4,266,026, 4,293,654, 4,301,249, 4,440,853 and 4,442,206.
As representative of the U.S. Patent art relating to organic polymeric porous microcapsules for encapsulating various biocatalysts mention can be made of the following: U.S. Pat. Nos. 3,522,346--Chang; 4,349,530--Roper; 4,353,888--Sefton; 4,148,689--Hino, et al; 4,310,554 Olson, et al; 3,767,790 and 3,860,490--Guttag; 4,431,428--Schmer; 4,321,327 Chen, et al; and the following group of patents, all assigned to Damon Corporation: U.S. Pat. Nos. 4,251,387, 4,255,411, 4,257,884, 4,322,311, 4,324,683, 4,352,883, 4,391,909, 4,407,957 and 4,409,331.
There is also a large body of patent art relating to the use of solid beads, which may or may not be porous, on which microorganisms and/or enzymes can be fixed or immobilized. For example, mention can be made of the following U.S. Patents: 4,343,901--DeFilippi; 4,189,534 and 4,293,654--Levine, et al; 4,153,510 Messing; 3,717,551--Bizzini, et al, and others. See also, K. Nilsson, et al., "Entrapment of animal cells for production of monoclonal antibodies and other biomolecules," Nature, Vol. 302, Apr. 14, 1983, pp. 629-630.
U.S. Pat. No. 3,875,008 to Yoshino, et al describes the encapsulation of enzymes and/or microorganisms in the lumen of a hollow semi-permeable polymeric filament which may be opened or closed at its ends.
The hollow fiber permselective membranes have the disadvantages of requiring headers which are difficult to manufacture and seal, frequent breakage, and especially for the organic fibers, inherently slow separation rates due to the requirement of having to use relatively thick separation membranes to obtain the necessary strength required to support the weight of the membranes. Moreover, the hollow fiber cell culture devices generally function primarily as supports and merely adhere the cells or other biocatalysts to the outer surface of the hollow fiber or in the pores of spongelike hollow-fibers or in the pores of solid porous microspheres or microbeads, and therefor do not provide a sufficiently sterile environment.
The hollow semipermeable organic microcapsules and gelatinous microbeads and the hollow semipermeable fibers which encapsulate cells, bacteria or other biocatalysts can provide an adequate sterile environment but suffer from the defect of inherently slow permeation rates due to the wall thickness of the membrane. However, even where the biocatalyst is encapsulated within the lumen of a hollow fiber, as in the above mentioned Yoshino, et al patent, there is still the risk of fiber breakage which can contaminate and render useless the entire or substantial portion of a production run. In addition, to date no practical economical method has been developed to fabricate the hollow microcapsule containers with uniform size or wall thickness and therefore control of the biotech processes using bioreactors based on these hollow permselective microcapsules is extremely difficult. Still further, these organic microcapsules and gels are generally fragile and cannot be stacked to any significant height, or used in, for example, a fluidized bed process, since the weight of the column or particle-to-particle or particle-to-wall contact can rupture, distort, or otherwise damage and ruin the microcapsule containers and gel beads.